Therapeutic applications of pro-apoptotic benzodiazepines

ABSTRACT

Benzodiazepine compounds, and methods for using those compounds are provided. Some of the benzodiazepine compounds include 1,4-benzodiazepine-2-one and 1,4-benzodiazepine-2,5-dione compounds of the following structures: wherein R&lt;SUB&gt;1&lt;/SUB&gt;, R&lt;SUB&gt;2&lt;/SUB&gt;, R&lt;SUB&gt;3 &lt;/SUB&gt;and R&lt;SUB&gt;4 &lt;/SUB&gt;are as defined. The invention also includes enantiomers, pharmaceutically acceptable salts, prodrugs or derivatives of the benzodiazepine compounds. Any one or more of these benzodiazepine compounds can be used to treat a variety of dysregulatory disorders related to cellular death. Such disorders include autoimmune disorders, inflammatory conditions, hyperproliferative conditions, viral infections, and atherosclerosis. In addition, the above compounds can be used to prepare medicaments to treat the above-described dysregulatory disorders. The benzodiazepines can also be used in drug screening assays and other diagnostic methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure claims priority to U.S. Provisional ApplicationNos. 60/191,855, filed Mar. 24, 2000; 60/131,761, filed Apr. 30, 1999;and 60/165,511, filed Nov. 15, 1999, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention is in the field of medicinal chemistry and relatesto other areas such as pharmacology, biochemistry and organic chemistry.In particular, it provides novel chemical compounds, and theirtherapeutic uses.

BACKGROUND

Multicellular organisms exert precise control over cell number. Thenormal structure and function of tissues depends on the maintenance ofappropriate cell numbers. A balance between cell proliferation and celldeath (White, E. (1996) Genes Dev. 10:1-15) achieves this homeostasis.

Cell death occurs in nearly every type of vertebrate cell via necrosisor through a suicidal form of cell death, known as apoptosis. Apoptosisis triggered by a variety of extracellular and intracellular signalsthat engage a common, genetically programmed death mechanism (Wyllie, A.H. (1995) Curr. Opin. Gen. Dev. 5:97-104). As a regulatory process,apoptosis influences diverse biological phenomena including thedevelopment of neural architecture, the immune system's ability todiscriminate between self and non-self, and the expunging of redundant,damaged or infected cells.

It has become evident that many diseases are associated withdysregulation of the process of cell death. Experimental models haveestablished a cause-effect relationship between derangement in themechanism regulating apoptosis or necrosis and the pathenogenicity ofvarious neoplastic, autoimmune and viral diseases (Thompson, C. B.(1995) Science 267:1456-1462). A well-defined example is the effect ofaberrant, high-level expression of bcl-2 on lymphoma development. Thebcl-2 oncogene was originally identified as the genetic element locatedat the t(14:18) chromosomal translocation breakpoint present in manyB-cell follicular lymphomas (Korsmeyer, S. J. (1992) Blood 359:554-556).Since that discovery, it has been convincingly established that thebcl-2 gene product inhibits apoptosis induced by a variety of stimuliand that its oncogenic potential stems from its ability to derailapoptosis (Sentman, C. L. et al. (1994) Cell 67:878-888; and McDonnell,T. J. and Korsmeyer, S. J. (1991) Nature 349:254-256).

Failed or reduced apoptosis is associated with the development of humanautoimmune lymphoproliferative syndrome as well as mouse models of thisdisease. MRL-lpr or gld mice develop lymphadenopathy, splenomegaly,nephritis and arthritis; as well they produce large quantities ofautoantibodies (Cohen, P. L. and Eisenberg, R. A. (1991) Annu. Rev.Immunol. 9:243-269).

These mice carry loss of function mutations in the genes encoding FASand Fas ligand, respectively (Adachi, M. et al. (1993) Proc. Natl. Acad.Sci. USA 90:1756-1760); Takakashi, T. et al. (1994) Cell 76:969-976).FAS, a ubiquitously expressed cell surface receptor, normally generatesan apoptotic response upon binding with Fas ligand (Itoh, N. et al.(1991) Cell 66:233-243). In mice carrying these loss of functionmutations, the disruption of FAS signaling renders T cells resistant toperipheral deletion by apoptosis (Russell, H. et al. Proc. Natl. Acad.Sci. USA 90:4409-4413). The inappropriate survival of these cellsresults in a pathologic accumulation of T and B cells evidenced by theneoplastic-like growth of lymphoid tissues and high-level autoantibodyproduction. In humans, autoimmune lymphoproliferative syndrome sharessimilarities with the mouse phenotype including lymphadenopathy,splenomegaly, autoantibodies and autoimmune manifestations. Patientswith this disease likewise carry mutation in the FAS gene (Nagata, S.(1998) J. Hum. Genet. 43:2-8).

Benzodiazepine compounds have been traditionally known to bind tobenzodiazepine receptors in the central nervous system (CNS) and thushave been used to treat various CNS disorders including anxiety andepilepsy. More recently, peripheral benzodiazepine receptors have alsobeen identified, which receptors may incidentally also be present in theCNS. Benzodiazepines and related structures have pro-apoptotic andcytotoxic properties useful in the treatment of transformed cells grownin tissue culture. There is therapeutic potential for this class ofagents against cancer and other neoplastic diseases. Two specificexamples shown are neuroblastoma and ovarian cancer.

Neuroblastoma is the most common extracranial solid tumor found inchildren. Modern treatments, which include chemotherapy, radiationtherapy and surgery, have not significantly reduced the mortality ofmetastatic neuroblastoma. Novel therapies are needed to improve survivalof children with this disease. We show that benzodiazepine compounds areable to slow the growth of these tumor cells. See, Sugimoto, T. et al.(1984) J. Natl. Cancer Inst. 73:51-57; Schwab, M. et al. (Sep. 15, 1983)Nature 305:245-248; and Dive, C. and Wyllie, A. H. (1993) Apoptosis andCancer Chemotherapy, Oxford Blackwell, pp. 21-56.

Ovarian cancer is difficult to treat due to chemoresistance shown by thepatient to standard chemotherapy drugs. Treatment failures are usuallyattributed to the emergence of chemotherapy resistant cells. We showthat benzdiazepine compounds are able to kill ovarian cancer cells thatare chemoresistant. See, Pestell, K. E. et al. (1998) Int. J. Cancer77(6):913-918; Beale, P. J. et al. (2000) Br. J. Cancer 82(2):436-440;Ozols, R. F. (February 1999) Semin. Oncol. 26(1 Suppl. 2):84-89; Liu, J.R. et al. (September 1998) Gynecol. Oncol. 70(3):398-403; Chumakov, A.M. et al. (November 1993) Oncogene 8(11):3005-3011; and Raynaud, F. I.et al. (August 1996) Br. J. Cancer 74(3):380-386.

Several benzodiazepine analogs have been reported as analgesic andanti-inflammatory agents. See, for example, U.S. Pat. Nos. 4,076,823,4,110,337, 4,495,101, 4,751,223 and 5,776,946.

U.S. Pat. Nos. 5,324,726 and 5,597,915 disclose that somebenzodiazepines are antagonists of cholecystokinin and gastrin and thusmight be useful to treat certain gastrointestinal disorders.

Certain benzodiazepines have also been explored as inhibitors of humanneutrophil elastase and thus potentially useful to treat the humanneutrophil elastase-mediated conditions such as myocardial ischemia,septic shock syndrome, among others. See U.S. Pat. No. 5,861,380.

U.S. Pat. No. 5,041,438 reported that certain benzodiazepines could beuseful as anti-retroviral agents. However, as it will become apparentfrom the description below, the present invention provides novel methodsand compositions that are distinct from the above-disclosed methods andcompositions.

DISCLOSURE OF THE INVENTION

This invention provides methods for treating a condition associated withdysregulation of the process of cell death in a subject, comprisingadministering to the subject an effective amount of a benzodiazepinecompound. In one aspect, the benzodiazepines of the class are identifiedby their inability to bind to a central benzodiazepine receptor or withlow affinity to a peripheral benzodiazepine receptor. In a furtheraspect, the benzodiazepines of the class are identified by having theability to induce cell death under condition of low serum as defined,infra. In a further aspect, the class of benzodiazepines are identifiedby having both of these above-noted characteristics. In a yet furtheraspect of this invention, chronic inflammatory conditions arespecifically excluded from the group of conditions that can be treatedby any class or specific compounds of the class of benzodiazepinesidentified herein.

In one aspect, the benzodiazepine compounds have the structure:

or its enantiomer, wherein, R₁ is aliphatic or aryl;

R₂ is aliphatic, aryl, —NH₂, —NHC(═O)—R₅, or a moiety that participatesin hydrogen bond formation,

wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇,wherein R₆ is an aliphatic linker of 1-6 carbons and R₇ is aliphatic,aryl, or heterocyclic; and

each of R₃ and R₄ is independently hydrogen, hydroxy, alkoxy, halo,amino, lower-alkyl-substituted-amino, acylamino, hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, aryl, orheteroaryl;

or a pharmaceutically acceptable salt, prodrug or derivative thereof.

The cell death can be induced by necrosis, apoptosis or regulation ofthe FAS pathway. The conditions associated with the dysregulation of aprocess of cell death include but are not limited to: autoimmunediseases such as systemic lupus erythematosus, rheumatoid arthritis,Sjögren's syndrome, graft-versus-host-disease, and myasthenia gravis;chronic inflammatory conditions such as psoriasis, asthma, and Crohn'sdisease; hyperproliferative disorders or neoplasms such as a B-cell or aT-cell lymphomas; and other conditions such as osteoarthritis andatherosclerosis. Methods are also provided for using the benzodiazepinecompounds to treat the conditions associated with the dysregulation ofcell death, wherein the condition is induced by a viral infection. Inaddition, in some aspects, methods are provided to treat a viralinfection by using the benzodiazepines of the present invention.

Methods are also provided to co-administer one or more additional agentswith the benzodiazepines of the present invention, wherein suchadditional agents may include antineoplastic agents, immunosuppressants,anti-inflammatory agents, antiviral agents, or radiation.

The cell death to be achieved by the methods and compositions of thisinvention involve the cell or cells present in a tissue that are:autoimmunogenic or affected by an autoimmune disorder; inflammatory oraffected by inflammation; hyperproliferative; viral-infected;atherosclerosed or osteoarthritic.

Assay and diagnostic methods are also provided to identify agents usefulto treat a condition associated with dysregulation of the process ofcell death in a subject wherein the ability of a potential candidateagent to induce cell death is assayed by contacting the dysregulatedcell with a benzodiazepine compound. The assay includes maintaining thesuitable cell or tissue preferably in a low serum.

Methods are also presented to prepare medicaments to treat a conditionassociated with dysregulation of the process of cell death in a subject,wherein the conditions, the affected cells or tissue and thebenzodiazepine compounds are described as above. The invention alsoprovides novel 1,4-benzodiazepine compounds having the structure:

or its enantiomer,

wherein,

R₁ is aliphatic or aryl;

R₂ is —NHC(═O)—R₅,

wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇,wherein R₆ is an aliphatic linker of 1-6 carbons and R₇ is aliphatic,aryl, or heterocyclic; and

each of R₃ and R₄ is independently hydrogen, hydroxy, alkoxy, halo,amino, lower-alkyl-substituted-amino, acylamino, hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, aryl, orheterocyclic; or a pharmaceutically acceptable salt, prodrug orderivative thereof.

These various methods, uses, and compositions are further described ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a general synthetic scheme for solid-phase synthesis of1,4-benzodiazepine 2-one compounds of the present invention.

FIG. 2 shows a general synthetic scheme for solid-phase synthesis of1,4-benzodiazepine-2,5-dione compounds of the present invention.

FIG. 3 depicts disease progression analysis for MRL-lpr mice treatedaccording to the methods described herein (solid line) as compared tocontrols (dotted line). The percentage of disease-free animals (y-axis)is plotted over time (x-axis).

FIGS. 4A-4C depict footpad swelling in MRL-lpr mice treated according tothe methods described herein (FIG. 4A) as compared to controls (FIG.4B). FIG. 4C is a graphical analysis.

FIG. 5 shows the structure of Compound 1 (Bz-423).

FIGS. 6A-6B are graphs depicting the in vitro treatment of NZB/W Csplenocytes. FIG. 6A shows dose-response lymphotoxicity of Compound 1.Relative viability expresses viable cells after treatment as apercentage of viable control cells to allow meaningful comparisonbetween multiple experiments. FIG. 6B shows a comparison of Compound 1to other ligands of benzodiazepine receptors and protection againstCompound 1 killing by CsA. Compounds were compared at 10 and 20 μM, 10μM results shown. At 20 μM, only Compound 1 demonstrated any cytotoxiceffect (data not shown).

FIG. 7 depicts the in vitro analysis of cell populations. Experimentalconditions are identical to FIG. 6. Solid bars represent the percentageof viable cells staining with B220 (B-cells) after treatment with theindicated agent. Hatched bars represent cells staining with Thy1.2(T-cells).

FIG. 8 is a series of photographs showing representative glomeruli fromNZB/W mice. Panel 1—H&E, panel 2—IgG deposition, and panel 3—complementC3 deposition. All sections were photographed at 400×.

FIG. 9 shows that treatment with Compound 1 prevents the development ofautoimmune glomerulonephritis. H&E stained kidney sections were analyzedfor degree of nephritis using a 0-4+ scale. Solid bars represent micewith disease (>2+) at time of sacrifice, while hatched bars representhealthy mice (<2+); p<0.003.

FIG. 10 is a photograph of representative spleen sections from NZB/Wmice. Panel 1—low power (50×) H&E sections indicating a decrease inlymphoid content in treatment mice. Panel 2—medium power (200×) sectionsindicating increased numbers and clustering of TUNEL positive-cells inthe treatment mice. Panel 3—high power (400×) sections co-stained forTUNEL-positive cells (green) and B220 (red) indicating increased numbersof B220⁺ TUNEL positive cells in the treatment mice.

FIG. 11 is a bar graph depicting the efficacy of using benzodiazepine tokill D2 neuroblastoma cells in vitro

FIG. 12 is a graph that shows that 2B1 and SKOV3 cells are resistant toCDDP.

FIG. 13 is a graph that shows that ovarian cancer cells are killed byapplication of benzodiazepine in vitro.

MODES FOR CARRYING OUT THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to describe more fully the state of the art to which thisinvention pertains.

A. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“MOLECULAR CLONING: A LABORATORY MANUAL” Second Edition (Sambrook etal., 1989); “OLIGONUCLEOTIDE SYNTHESIS” (M. J. Gait, ed., 1984); “ANIMALCELL CULTURE” (R. I. Freshney, ed., 1987); the series “METHODS INENZYMOLOGY” (Academic Press, Inc.); “HANDBOOK OF EXPERIMENTALIMMUNOLOGY” (D. M. Weir & C. C. Blackwell, eds.); “GENE TRANSFER VECTORSFOR MAMMALIAN CELLS” (J. M. Miller & M. P. Calos, eds., 1987); “CURRENTPROTOCOLS IN MOLECULAR BIOLOGY” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: THE POLYMERASE CHAIN REACTION” (Mullis et al.,eds., 1994); and “CURRENT PROTOCOLS IN IMMUNOLOGY” (J. E. Coligan etal., eds., 1991).

B. Definitions

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. Similarly, use of “a compound” for treatmentor preparation of medicaments as described herein contemplates using oneor more compounds of this invention for such treatment or preparationunless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

A “benzodiazepine” refers to a seven membered non-aromatic heterocyclicring fused to a phenyl ring wherein the seven-membered ring has twonitrogen atoms, as part of the heterocyclic ring. In some aspects, thetwo nitrogen atoms are in 1 and 4 positions, as shown in the generalstructure below.

The benzodiazepine can be substituted with one keto group (typically atthe 2-position), or with two keto groups (one each at the 2- and 5positions). When the benzodiazepine has two keto groups (i.e., one eachat the 2 and 5 positions), it is referred to asbenzodiazepine-2,5-dione. Most generally, the benzodiazepine is furthersubstituted either on the six-membered phenyl ring or on theseven-membered heterocyclic ring or on both rings by a variety ofsubstituents. These substituents are described more fully below.

The term “dysregulation of the process of cell death” is intended toencompass any aberration in the ability of (or predisposition of) a cellto undergo cell death via either necrosis or apoptosis. Dysregulation ofcell death is associated with or induced by a variety of conditions,including for example, autoimmune disorders (e.g., systemic lupuserythematosus, rheumatoid arthritis, graft-versus-host disease,myasthenia gravis, Sjögren's syndrome, etc.), chronic inflammatoryconditions (e.g., psoriasis, asthma and Crohn's disease),hyperproliferative disorders (e.g., tumors, B cell lymphomas, T celllymphomas, etc.), viral infections (e.g., herpes, papilloma, HIV), andother conditions such as osteoarthritis and atherosclerosis.

It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

An “autoimmune disorder” is any condition in which an organism producesantibodies or immune cells which recognize the organism's own molecules,cells or tissues. Non-limiting examples of autoimmune disorders includerheumatoid arthritis, Sjögren's syndrome, graft versus host disease,myasthenia gravis, and systemic lupus erythematosus.

A “hyperproliferative disorder” is any condition in which a localizedpopulation of proliferating cells in an animal is not governed by theusual limitation of normal growth. Examples of hyperproliferativedisorders include tumors, neoplasms, lymphomas and the like. A neoplasmis said to be benign if it does not undergo, invasion or metastasis andmalignant if it does either of these. A metastatic cell or tissue meansthat the cell can invade and destroy neighboring body structures.Hyperplasia is a form of cell proliferation involving an increase incell number in a tissue or organ, without significant alteration instructure or function. Metaplasia is a form of controlled cell growth inwhich one type of fully differentiated cell substitutes for another typeof differentiated cell. Metaplasia can occur in epithelial or connectivetissue cells. A typical metaplasia involves a somewhat disorderlymetaplastic epithelium.

A “chronic inflammatory condition” shall mean those conditions that arecharacterized by a persistent inflammatory response with pathologicsequelae. This state is characterized by infiltration of mononuclearcells, proliferation of fibroblasts and small blood vessels, increasedconnective tissue, and tissue destruction. Examples of chronicinflammatory diseases include Crohn's disease, psoriasis, and asthma.Autoimmune diseases such as rheumatoid arthritis and systemic lupuserythematosus can also result in a chronic inflammatory state.

The “co-administration” refers to administration of more than one agentor therapy to a subject. By “sensitizing agent” is meant any agent whichincreases the sensitivity of a target cell or tissue to othertherapeutic agents. In the context of the present invention,co-administration of the claimed compounds results in a surprising andunexpected therapeutic effect, particularly in the treatment ofconditions in which apoptosis or necrosis is dysregulated. The compoundsdescribed herein also appear to sensitize target cells to thetherapeutic actions of other agents.

Co-administration may be concurrent or, alternatively, the chemicalcompounds described herein may be administered in advance of orfollowing the administration of the other agent(s). The appropriatedosage for co-administration can be readily determined by one skilled inthe art. When co-administered with another therapeutic agent, both theagents may be used at lower dosages. Thus, co-administration isespecially desirable where the claimed compounds are used to lower therequisite dosage of known toxic agents. “Toxic” refers to anydetrimental or harmful effects on a cell or tissue.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a solid support, adetectable agent or label) or active, such as an adjuvant.

As used herein, “solid phase support” or “solid support,” usedinterchangeably, is not limited to a specific type of support. Rather alarge number of supports are available and are known to one of ordinaryskill in the art. Solid phase supports include silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels. As used herein, “solid support” also includes syntheticantigen-presenting matrices, cells, and liposomes. A suitable solidphase support may be selected on the basis of desired end use andsuitability for various protocols. For example, for peptide synthesis,solid phase support may refer to resins such as polystyrene (e.g.,PAM-resin obtained from Bachem, Inc., Peninsula Laboratories, etc.),POLYHIPE®) resin (obtained from Aminotech, Canada), polyamide resin(obtained from Peninsula Laboratories), polystyrene resin grafted withpolyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) orpolydimethylacrylamide resin (obtained from Milligen/Biosearch,California).

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'SPHARMACEUTICAL SCIENCES, 15th Ed., Mack Publ. Co., Easton, Pa. (1975).

“Pharmaceutically acceptable salt, prodrug or derivative” as usedherein, relate to any pharmaceutically acceptable salt, ester, ether,salt of an ester, solvate, such as ethanolate, or other derivative of acompound of the present invention which, upon administration to arecipient, is capable of providing (directly or indirectly in the caseof a prodrug) a compound of this invention or an active metabolite orresidue thereof. Particularly favored derivatives and prodrugs are thosethat increase the bioavailability of the compounds of this inventionwhen such compounds are administered to a mammal (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system).

As is known to those of skill in the art, “salts” of the compounds ofthe present invention may be derived from inorganic or organic acids andbases. Examples of acids include hydrochloric, hydrobromic, sulfuric,nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic,salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,methanesulfonic, ethanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic and benzenesulfonic acids. Other acids, such asoxalic, while not in themselves pharmaceutically acceptable, may beemployed in the preparation of salts useful as intermediates inobtaining the compounds of the invention and their pharmaceuticallyacceptable acid addition salts.

Examples of bases include alkali metal (e.g., sodium) hydroxides,alkaline earth metal (e.g., magnesium) hydroxides, ammonia, andcompounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl. Examples of saltsinclude: acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺(wherein W is a C₁₋₄ alkyl group).

For therapeutic use, salts of the compounds of the present inventionwill be pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

The term “derivative” of a compound as used herein, means a chemicallymodified compound wherein the chemical modification takes place eitherat a functional group of the compound or on the aromatic ring.Non-limiting examples of 1,4-benzodiazepine derivatives of the presentinvention may include N-acetyl, N-methyl, N-hydroxy groups at any of theavailable nitrogens in the compound. Additional derivatives may includethose having a trifluoromethyl group on the phenyl ring.

C. Methods of Treatment

The therapeutic potential for using benzodiazepines for theirpro-apoptotic and cytotoxic properties is great. The class of agents iseffective in treating cancers and other neoplastic diseases. As notedabove, the present invention provides methods of treating conditionsthat are, in one embodiment, related in that they arise as the result ofdysregulation of the normal process of cell death in the cells or tissueof a subject.

For the purpose of illustration only, such conditions include, but arenot limited to autoimmune disorders (e.g., systemic lupus erythematosus,rheumatoid arthritis, graft-versus-host disease, Sjögren's syndrome andmyasthenia gravis); hyperproliferative disorders, (such as—B or T celllymphoma, neuroblastoma, and chronic lymphocytic leukemia); chronicinflammatory conditions (such as psoriasis, asthma, or Crohn's disease);other conditions such as osteoarthritis and atherosclerosis; and thoseinduced by DNA and/or RNA viral infections, wherein the viruses includebut are not limited to, herpes virus, papilloma virus and humanimmunodeficiency virus (HIV).

These disorders are treated by administering an effective amount of thebenzodiazepine compounds described herein. The various benzodiazepinecompounds are described more fully below. These compounds aretherapeutically effective on their own, and have few or no toxic effectswhen administered in large doses. Further, as described in detail below,co-administration of these compounds with other agents provides anunexpected synergistic therapeutic benefit. In the co-administrationmethods, the claimed compounds are also useful in reducing deleteriousside-effects of known therapeutic agents by decreasing the amount whichmust be administered to the subject.

The conditions which benefit from treatment with the compounds describedherein appear to share the common etiology of dysregulation of theprocess of cell death. As described above in the Background, normalapoptosis occurs via several pathways, with each pathway having multiplesteps. The methods described herein are useful in treating dysregulatedapoptosis regardless of the pathway or the step in the pathway where thedysfunction is occurring. In one embodiment, the condition is caused asthe result of dysregulation of the FAS apoptotic pathway. Similarly, thecompounds are also useful in treating dysregulated necrosis regardlessof the pathway or the step in the pathway where the dysfunction isoccurring.

Dysregulation of the process of cell death is associated with manyconditions. In neoplasms, for example, normal cell death is inhibited,allowing hyperproliferative growth of cells. Aberrant functioning ofthis process can also result in serious pathologies including autoimmunedisorders, viral infections, conditions induced by viral infections,neurodegenerative disease, and the like. The present invention providesmethods of treating these and other conditions. Without being bound byone theory, it seems that the effective compounds described hereininduce or promote cell death in when this process is malfunctioning.Thus, in addition to treating conditions associated with dysregulationof apoptosis, the compounds of this invention also treat conditions inwhich there may not be any apoptotic defect. For example, in certainviral infections, while there may not be any apoptotic defect, celldeath may be promoted by inducing necrosis.

The condition to be treated is generally determined by noting thepresence of symptoms in the subject or by noting phenotypic or genotypicchanges in the cells of the subject, in particular, the inability of thecell to undergo apoptosis or necrosis. Phenotypic changes associatedwith the neoplastic state of a cell (a set of in vitro characteristicsassociated with a tumorigenic ability in vivo) include more rounded cellmorphology, looser substratum attachment, loss of contact inhibition,loss of anchorage dependence, release of proteases, increased sugartransport, decreased serum requirement, expression of fetal antigens,etc. See, Luria, et al., (1978) GENERAL VIROLOGY, 3rd edition, pp.436-446 (John Wiley & Sons, New York). To “treat” as intended herein,means to induce cell death (wherein the cell death is either apoptoticor necrotic) in cells or tissue which are causative (primary or distal)of the disorder being treated. For example, in hyperproliferativedisorders, the method will treat the disorder by inducing apoptosis ofthe hyperproliferative cells, such as neoplastic cells. In thisembodiment, reduction in tumor size or tumor burden is one means toidentify that the object of the method has been met. In other aspects,“to treat” encompasses restoration of immune function or regulation ofimmune dysfunction, as in autoimmune disorders and chronic inflammatoryconditions. In other aspects, viral titer is eliminated or reduced inthe subject being treated. In further aspects, “to treat” means toameliorate the symptoms associated with a particular disease, e.g.,cachexia in cancer or HIV infection or inflammation in arthritis. Instill further aspects, prophylactic as well as therapeutic use of thecompounds and methods of this invention are intended.

In the embodiment wherein the condition being treated is an autoimmunedisease, the use of the methods disclosed herein will reduceautoantibody production and lead to a decrease in inflammation andtissue destruction. Thus, the cell that is being treated may be the cellthat itself is autoimmunogenic or is affected distally by an autoimmunereaction, wherein it is desirable to induce cell death in such cells ortissue containing such cells. Similarly, in the case of treatinginflammatory conditions, the cell that is being treated may be theinflammatory cell itself or is distally affected by inflammation whereinit is desirable to induce cell death in such cells or tissue containingsuch cells and thus reduce inflammation.

In a further embodiment, the cell being treated is a virally infectedcell or a cell or tissue that previously has been infected. Successfultherapy induces cell death and therefore a reduction in viral titer.This result is easily determined by assaying viral titer or by noting areduction in cell number. It should be noted that, as can be inferredfrom the statements, supra, it may be desirable to induce cell deatheven among those cells that do not have any viral remnants or othersigns of viral infections at the time of treatment because a viralinfection that occurred much earlier in time could still causedisruption of cell death at a much later time.

Cell death may be assayed as described herein and in the art. Cell linesare maintained in appropriate culture conditions (e.g., gas (CO₂),temperature and media) and for an appropriate amount of time to attainexponential proliferation without density dependent constraints. Cellnumber and or viability may be measured using standard techniques, suchas trypan blue exclusion/hemo-cytometry, or MTT dye conversion assay.Alternatively, the cell may be analyzed for the expression of genes orgene products associated with aberrations in apoptosis or necrosis.

It should be understood that the various dysregulatory disordersdescribed above can be treated by any one or more of the benzodiazepinecompounds, including the many alternative and specific embodimentspresented herein. These benzodiazepines are described more fully below.

Antiviral Activity

In another embodiment, the compounds of the present invention may haveantiviral activity independent of their efficacy to induce cell death.One aspect or method for inhibiting viral replication and/or propagationcomprises contacting the virus with an effective amount of one or morecompounds and/or compositions of the present invention. The contactingis conducted under suitable conditions to inhibit viral replicationand/or propagation. In another aspect, the method comprises preventingviral infection and/or propagation in a cell or tissue by contacting thecell or tissue with an effective amount of the compounds and/orcompositions as defined above. The contacting is conducted undersuitable conditions to such that viral infection and/or propagation isinhibited.

The inhibition or prevention of viral infection, replication and/orpropagation can be measured by assaying for viral titer, which methodsare well known to those of skill in the art. By inhibiting and reducingviral replication and proliferation, viral infectivity also is inhibitedand reduced and the host cells are suitably treated for viral infectionwith the additional benefit that associated pathologies also aretreated.

As used herein, the term “suitable conditions” includes in vitro, exvivo or in vivo conditions. These terms are well-known in the art.

The viruses that are contemplated under the present methods include RNAand/or DNA viruses. By way of example only, such viruses are of herpes,non-herpes and retroviral origins. Major examples of human pathogens ofthe herpes virus family include herpes simplex viruses (HSV) 1, 2, andcercopithecine herpes virus 1 (B-virus); varicella-zoster, Epstein-Barrvirus (EBV); Lymphocryptovirus; human herpesviruses 6-8 (HHV6-S);kaposi-associated herpes virus (KHV); herpesvirus simiae, and humancytomegalovirus (HCMV). See, for example, Gallant, J. E. et al., J.Infect. Dis. 166:1223-1227, (1992).

Animal pathogens of herpesviral origin include infectious bovinerhinotracheitis virus, bovine mammillitis virus, and cercopithecineherpesvirus (B-virus), among others.

The human viruses of non-herpes origin include influenza viruses A, Band C; parainfluenza viruses—1, 2, 3 and 4; adenovirus; reovirus;respiratory syncytial virus; rhinovirus; coxsackle virus; echo virus;rubeola virus; hepatitis viruses of the types B and C (HBV and HCV); andpapovavirus.

The animal viruses of non-herpes origin include pseudorabies virus (PRV,of swine), equine rhinopneumonitis, coital exanthema viruses (varicellaviruses); lymphocryptovirus; Marek's disease virus, Bovine Herpesvirus-1(BHV-1), herpesvirus Pseudorabies virus (PRV).

The viruses of retroviral origin that are contemplated to be treatableby the compounds and compositions of this invention include humanimmunodeficiency viruses (HIV) of the types 1 and 2 and humanlymphotropic 1 and 2 viruses (HTLV-I and II).

D. Methods of Identifying Potential Therapeutic Agents

Also provided herein is an assay to identify a potential agent to treata condition associated with the dysregulation of the apoptotic ornecrotic pathway. The method comprises contacting the dysregulated cell,i.e., a cell affected by the disorder (e.g. a tumor cell when thecondition is hyperproliferative) or an immune cell (a neutrophil,basophil, eosinophil, monocyte, or lymphocyte) when the condition is achronic inflammatory condition or an autoimmune disorder) with theagent. In a further aspect of this invention, a control cell is furtherassayed with or without a benzodiazepine compound. The benzodiazepinecompound may be a 1,4-benzodizepine compound as described herein. Celldeath as compared to the control cell is also noted and compared. Toidentify these potential therapeutic agents, appropriate assayconditions (e.g., incubation time, temperature, culture maintenancemedium, etc.) can be readily determined by one of skill in the art.Serum may be obtained from any commercial source, for example, fetalbovine serum from Gibco BRL (Gaithersburg, Md.). The cells are culturedwith the test agent for a sufficient amount of time. Following theappropriate incubation period, cell death may then be assayed by anymeans described above, for example by MTT dye trypan exclusion. Thus,novel cytotoxic agents can be identified by their ability to induce celldeath to the dysregulated cells. Further, comparisons can also be madeto cell death in control cells.

The present inventors have discovered that when the cells are maintainedin low serum conditions, cytotoxicity is greatly exacerbated and theincubation time is reduced to about 2 hours or less. This is quite anunexpected result since under standard incubation conditions, whichemploy higher serum levels, the required incubation time is oftenseveral hours, approaching in some cases 24 hours or more. “Low serum”as used herein refers to culture media containing less than about 10%per volume down to or equal to less than about 0.1% (v/v). It-should beunderstood that within this range the concentration is flexible, and theapplicants contemplate any possible subrange in increments of about 0.1%within this range, for example, less than or equal to about any of 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, . . . 1.0, . . . 1.5, . . . 2.0, . .. 2.5, . . . 3.0, . . . 3.5, . . . 5%, etc., to about 9.9, and to about10% of serum (% v/v).

Thus, in one aspect, the benzodiazepines of this invention induceapoptosis in low serum as defined above.

In a further aspect, the benzodiazepines of this invention are furthercharacterized and identified by their lack of ability to bind to acentral benzodiazepine receptor or to bind with low affinity to aperipheral benzodiazepine receptor. These compounds can be identified byusing methods well-known in the art.

For example, the binding affinity of a benzodiazepine compound for aperipheral benzodiazepine receptor can be determined according towell-established methodology as described in Schoemaker, H. et al.(1983) J. Pharm. Exp Ther. 225:61-69; and Doble, is A. et al. (1987)Brain Res. Bull. 18:49-61.

Briefly, the method comprises comparing the potency of a benzodiazepinecompound with that of a well-known high affinity binding agent such as1-(2-chlorophenyl)-N-methyl-N-(-1,methylpropyl)-3-isoquinolinecarboxamide(PK11195), wherein the ability of the benzodiazepine compound todisplace PK511195 from the peripheral benzodiazepine receptors in acompetitive binding assay.

In any of the above assay methods, the benzodiazepine compound can bedetectably labeled. Such detectable labeling includes labeling with anisotope or with a fluorescing moiety. Examples of isotope labelinginclude usage of stable or radioactive isotopes of one or more atoms onthe benzodiazepine molecule.

Methods for introducing such detectable labels and for detecting thelabels are well-known in the art. For example, the radioisotope labelcan be detected using special instrumentation, including electron spinresonance spectrometers. The stable isotopes can be detected using massspectrometers, or magnetic resonance spectrometers. The fluorescentlabels can be detected using fluorescent spectrometers. Many of theseinstruments are commercially available and their operation is within theordinary skill in the art.

The benzodiazepine compounds that can be used in the assay anddiagnostic methods are described in greater detail below. It should beunderstood that all the compounds described therein, including the manygeneral and specific embodiments, can be used in the assay anddiagnostic methodology.

E. Use of Benzodiazepine Compounds for Preparing Medicaments

The benzodiazepine compounds of the present invention are also useful inthe preparation of medicaments to treat a variety of conditionsassociated with dysregulation of cell death as described above. Inaddition, the compounds can also be used to prepare medicaments fortreating other disorders wherein the effectiveness of thebenzodiazepines are known or predicted. Such disorders may includeneurological or neuromuscular disorders. The methods and techniques forpreparing medicaments of a compound are well-known in the art. Somepossible pharmaceutical formulations and routes of delivery are detailedbelow.

Thus, one of skill in the art would readily appreciate that any one ormore of the compounds described more fully below, including the manyspecific embodiments, can be used by applying standard pharmaceuticalmanufacturing procedures to prepare medicaments to treat the manydisorders described herein above. Such medicaments can be delivered tothe subject by using delivery methods that are well-known in thepharmaceutical arts.

F. Compositions and Formulations

While it is possible for the agent to be administered alone, it ispreferable to present it as a pharmaceutical formulation comprising atleast one active ingredient, as defined above, together with a solidsupport or alternatively, together with one or more pharmaceuticallyacceptable carriers therefor and optionally other therapeutic agents.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient.

Formulations include those suitable for oral, rectal, nasal, topical(including transdermal, buccal and sublingual), vaginal, parenteral(including subcutaneous, intramuscular, intravenous and intradermal) andpulmonary administration. The formulations may conveniently be presentedin unit dosage form and may be prepared by any methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general, the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) surface-active or dispersing agent. Moldedtablets maybe made by molding in a suitable. machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention may be formulated as an ointment, cream, suspension,lotion, powder, solution, paste, gel, spray, aerosol or oil.Alternatively, a formulation may comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients, andoptionally one or more excipients or diluents.

If desired, the aqueous phase of the cream base may include, forexample, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the agent throughthe skin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulfoxide (DMSO) and related analogues.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in an known manner. While this phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and/or fat make up the so-called emulsifyingointment base which forms the oily dispersed phase of the creamformulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus the cream should preferably be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the agent, such carriers as are known in theart to be appropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebulizer, include aqueous or oily solutions of the agent.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampoules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies.

G. Pharmaceutical Delivery

Various delivery systems are known and can be used to administer atherapeutic agent of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis (see, e.g.,Wu and Wu, (1987), J. Biol. Chem. 262:4429-4432), and the like. Methodsof delivery include but are not limited to, intra-arterial,intra-muscular, intravenous, intranasal, and oral routes. In a specificembodiment, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, by injection, or by means of a catheter.

The agents identified herein as effective for their intended purpose canbe administered to subjects or individuals susceptible to or at risk ofdeveloping a condition correlated with dysregulation of the apoptotic ornecrotic pathway. When the agent is administered to a subject such as amouse, a rat or a human patient, the agent can be added to apharmaceutically acceptable carrier and systemically or topicallyadministered to the subject. To determine patients that can bebeneficially treated, a tissue sample is removed from the patient andthe cells are assayed for sensitivity to the agent.

Therapeutic amounts can be empirically determined and will vary with thepathology being treated, the subject being treated and the efficacy andtoxicity of the agent. When delivered to an animal, the method is usefulto further confirm efficacy of the agent. One example of an animal modelis MLR/MpJ-lpr/lpr (“MLR-lpr”) (available from Jackson Laboratories, BalHarbor, Me. ). MLR-lpr mice develop systemic autoimmune disease.Alternatively, other animal models can be developed by inducing tumorgrowth, for example, by subcutaneously inoculating nude mice with about10⁵ to about 10⁹ hyperproliferative, cancer or target cells as definedherein. When the tumor is established, the compounds described hereinare administered, for example, by subcutaneous injection around thetumor. Tumor measurements to determine reduction of tumor size are madein two dimensions using venier calipers twice a week. Other animalmodels may also be employed as appropriate. Such animal models for theabove-described diseases and conditions are well-known in the art. See,for example, (1992) Am. J. Pathol. 36:875-882.

Administration in vivo can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with thecomposition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents canbe readily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to anagent of the present invention, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including oral, rectal, nasal, topical (includingtransdermal, aerosol, buccal and sublingual), vaginal, parental(including subcutaneous, intramuscular, intravenous and intradermal) andpulmonary. It will also be appreciated that the preferred route willvary with the condition and age of the recipient, and the disease beingtreated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or orally administered, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

H. Co-Administration

The present invention also includes methods involving co-administrationof the compounds described herein with one or more additional activeagents. Indeed, it is a further aspect of this invention to providemethods for enhancing prior art therapies and/or pharmaceuticalcompositions by co-administering a compound of this invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compounds describedherein are administered prior to the other active agent(s). Thepharmaceutical formulations and modes of administration may be any ofthose described above. In addition, the two or more co-administeredchemical agents, biological agents or radiation may each be administeredusing different modes or different formulations.

The agent or agents to be co-administered depends on the type ofcondition being treated. For example, when the condition being treatedis hyperproliferation, the additional agent can be a chemotherapeuticagent or radiation. When the condition being treated is an autoimmunedisorder, the additional agent can be an immunosuppressant or ananti-inflammatory agent. When the condition being treated is chronicinflammation, the additional agent can be an anti-inflammatory agent.When the condition being treated is a viral infection or conditionsinduced by a viral infection, the additional agent can be an antiviralagent. The additional agents to be co-administered, such as anticancer,immunosuppressant, anti-inflammatory, and antiviral agents can be any ofthe well-known agents in the art, including those that are currently inclinical use. The determination of appropriate type and dosage ofradiation treatment is also within the skill in the art or can bedetermined with relative ease.

Currently, treatment of the various conditions associated with abnormalapoptosis is limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. In certain cancers, for example, resistance to chemicals andradiation therapy has been shown to be associated with inhibition ofapoptosis. See, Desoize, B. (1994) Anticancer Res. 14:221-224.Similarly, some therapeutic agents have deleterious side effects,including non-specific lymphotoxicity, renal and bone marrow toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds described hereinwith the known agent. The compounds described herein appear to sensitizetarget cells to known agents and, accordingly, less of these agents areneeded to achieve a therapeutic benefit.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases weredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.Further, because the claimed compounds are themselves both effective andnon-toxic in large doses, co-administration of proportionally more ofthese compounds than known toxic therapeutics will achieve the desiredeffects while minimizing toxic effects.

I. The Benzodiazepine Compounds

The compounds of the present invention are benzodiazepine compounds. Insome aspects, the benzodiazepine compounds have the following structure:

or its enantiomer,

wherein,

R₁ is aliphatic or aryl;

R₂ is aliphatic, aryl, —NH₂, —NHC(═O)—R₅, or a moiety that participatesin hydrogen bonding,

wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇,wherein R₆ is an aliphatic linker of 1-6 carbons and R₇ is aliphatic,aryl, or heterocyclic,

each of R₃ and R₄ is independently a hydroxy, alkoxy, halo, amino,lower-alkyl-substituted-amino, acetylamino, hydroxyamino, an aliphaticgroup having 1-8 carbons and 1-20 hydrogens, aryl, or heterocyclic;

or a pharmaceutically acceptable salt, prodrug or derivative thereof.

In the above structures, R₁ is a hydrocarbyl group of 1-20 carbons and1-20 hydrogens. Preferably, R₁ has 1-15 carbons, and more preferably,has 1-12 carbons. Preferably, R₁ has 1-12 hydrogens, and morepreferably, 1-10 hydrogens. Thus R₁ can be an aliphatic group or an arylgroup.

The term “aliphatic” represents the groups commonly known as alkyl,alkenyl, alkynyl, alicyclic. The term “aryl” as used herein represents asingle aromatic ring such as a phenyl ring, or two or more aromaticrings that are connected to each other (e.g., bisphenyl) or fusedtogether (e.g., naphthalene or anthracene). The aryl group can beoptionally substituted with a lower aliphatic group (e.g., C₁-C₄ alkyl,alkenyl, alkynyl, or C₃-C₆ alicyclic). Additionally, the aliphatic andaryl groups can be further substituted by one or more functional groupssuch as —NH₂, —NHCOCH₃, —OH, lower alkoxy (C₁-C₄), halo (—F. —Cl, —Br,or —I). It is preferable that R₁ is primarily a nonpolar moiety.

In the above structures, R₂ can be aliphatic, aryl, —NH₂, —NHC(═O)—R₅,or a moiety that participates in hydrogen bonding, wherein R₅ is aryl,heterocyclic, R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇, wherein R₆ is analiphatic linker of 1-6 carbons and R₇ is an aliphatic, aryl, orheterocyclic. The terms “aliphatic” and “aryl” are as defined above.

The term “a moiety that participates in hydrogen bonding” as used hereinrepresents a group that can accept or donate a proton to form a hydrogenbond thereby. Some specific nonlimiting examples of moieties thatparticipate in hydrogen bonding include a fluoro, oxygen-containing andnitrogen-containing groups that are well-known in the art. Some examplesof oxygen-containing groups that participate in hydrogen bondinginclude: hydroxy, lower alkoxy, lower carbonyl, lower carboxyl, lowerethers and phenolic groups. The qualifier “lower” as used herein refersto lower aliphatic groups (C₁-C₄) to which the respectiveoxygen-containing functional group is attached. Thus, for example, theterm “lower carbonyl” refers to inter alia, formaldehyde, acetaldehyde.

Some nonlimiting examples of nitrogen-containing groups that participatein hydrogen bond formation include amino and amido groups. Additionally,groups containing both an oxygen and a nitrogen atom can alsoparticipate in hydrogen bond formation. Examples of such groups includenitro, N-hydroxy and nitrous groups.

It is also possible that the hydrogen-bond acceptor in the presentinvention can be the π electrons of an aromatic ring. However, thehydrogen bond participants of this invention do not include those groupscontaining metal atoms such as boron. Further the hydrogen bonds formedwithin the scope of practicing this invention do not include thoseformed between two hydrogens, known as “dihydrogen bonds.” See,Crabtree, R. H. (1998) Science 282:2000-2001, for further description ofsuch dihydrogen bonds.

The term “heterocyclic” represents a 3-6 membered aromatic ornonaromatic ring containing one or more heteroatoms. The heteroatoms canbe the same or different from each other. Preferably, at least one ofthe heteroatoms is nitrogen. Other heteroatoms that can be present onthe heterocyclic ring include oxygen and sulfur.

Aromatic and nonaromatic heterocyclic rings are well-known in the art.Some nonlimiting examples of aromatic heterocyclic rings includepyridine, pyrimidine, indole, purine, quinoline and isoquinoline.Nonlimiting examples of nonaromatic heterocyclic compounds includepiperidine, piperazine, morpholine, pyrrolidine and pyrazolidine.Examples of oxygen containing heterocyclic rings include, but notlimited to furan, oxirane, 2H-pyran, 4H-pyran, 2H-chromene, andbenzofuran. Examples of sulfur-containing heterocyclic rings include,but are not limited to, thiophene, benzothiophene, and parathiazine.

Examples of nitrogen containing rings include, but not limited to,pyrrole, pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,imidazolidine, pyridine, piperidine, pyrazine, piperazine, pyrimidine,indole, purine, benzimidazole, quinoline, isoquinoline, triazole, andtriazine.

Examples of heterocyclic rings containing two different heteroatomsinclude, but are not limited to, phenothiazine, morpholine,parathiazine, oxazine, oxazole, thiazine, and thiazole.

The heterocyclic ring is optionally further substituted with one or moregroups selected from aliphatic, nitro, acetyl (i.e., —C(═O)—CH₃), oraryl groups.

Each of R₃ and R₄ can be independently a hydroxy, alkoxy, halo, amino,or substituted amino (such as lower-alkyl-substituted-amino, oracetylamino or hydroxyamino), or an aliphatic group having 1-8 carbonsand 1-20 hydrogens. When each of R₃ and R₄ is an aliphatic group, it canbe further substituted with one or more functional groups such as ahydroxy, alkoxy, halo, amino or substituted amino groups as describedabove. The terms “aliphatic” is defined above. Alternatively, each of R₃and R₄ can be hydrogen.

It is well-known that many 1,4-benzodiazepines exist as optical isomersdue to the chirality introduced into the heterocyclic ring at the C₃position. The optical isomers are sometimes described as L- or D-isomersin the literature. Alternatively, the isomers are also referred to as R-and S-enantiomorphs. For the sake of simplicity, these isomers arereferred to as enantiomorphs or enantiomers. The 1,4-benzodiazepinecompounds described herein include their enantiomeric forms as well asracemic mixtures. Thus, the usage “benzodiazepine or its enantiomer”herein refers to the benzodiazepine as described or depicted, includingall its enantiomorphs as well as their racemic mixture.

From the above description, it is apparent that many specific examplesare represented by the generic formulas presented above. Thus, in oneexample, R₁ is aliphatic, R₂ is aliphatic, whereas in another example,R₁ is aryl and R₂ is a moiety that participates in hydrogen bondformation. Alternatively, R₁ can be aliphatic, and R₂ can be an—NHC(═O)—R₅, or a moiety that participates in hydrogen bonding, whereinR₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇, wherein R₆is an aliphatic linker of 1-6 carbons and R₇ is an aliphatic, aryl, orheterocyclic. A wide variety of such combinations arising from selectinga particular group at each substituent position are possible and allsuch combinations are within the scope of this invention.

Further, it should be understood that the numerical ranges giventhroughout this disclosure should be construed as a flexible range thatcontemplates any possible subrange within that range. For example, thedescription of a group having the range of 1-10 carbons would alsocontemplate a group possessing a subrange of, for example, 1-3, 1-5,1-8, or 2-3, 2-5, 2-8, 3-4, 3-5, 3-7, 3-9, 3-10, etc., carbons. Thus,the range 1-10 should be understood to represent the outer boundaries ofthe range within which many possible subranges are clearly contemplated.Additional examples contemplating ranges in other contexts can be foundthroughout this disclosure wherein such ranges include analogoussubranges within.

Some specific examples of the benzodiazepine compounds of this inventioninclude:

In summary, several benzodiazepine compounds are presented herein. Anyone or more of these benzodiazepine compounds can be used to treat avariety of dysregulatory disorders related to cellular death. Suchdisorders include autoimmune disorders, inflammatory conditions,hyperproliferative conditions, viral infections, and atherosclerosis. Inaddition, the above compounds can be used to prepare medicaments totreat the above-described dysregulatory disorders. The above-describedbenzodiazepines can also be used in drug screening assays and otherdiagnostic methods. The broad scope of the methodology, uses andcompositions described herein becomes readily apparent from acomprehensive reading of the entire description, noting that preferredand advantageous features of some aspects are applicable to otheraspects of the invention.

J. Preparation of Compounds

The above-described benzodiazepine compounds can be prepared usingeither solid-phase or soluble-phase combinatorial synthetic methods aswell as on an individual basis from well-established techniques. See,for example, Boojamra, C. G. et al. (1996) J. Org. Chem. 62:1240-1256;Bunin, B. A., et al. (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712;Stevens, S. Y. et al., (1996) J. Am. Chem. Soc. 118:10650-10651; Gordon,E. M., et al., (1994) J. Med. Chem. 37:51385-1401; and U.S. Pat. Nos.4,110,337 and 4,076,823, which are all incorporated by reference herein.For illustration, the following general methodologies are provided.

1. Preparation of 1.4-benzodiazepine-2-one Compounds

Improved solid-phase synthetic methods for the preparation of a varietyof 1,4-benzodiazepine-2-one derivatives with very high overall yieldshave been reported in the literature. See, for example, Bunin and Ellman((1992) J. Am. Chem. Soc. 114:10997-10998). Using these improvedmethods, the 1,4-benzodiazepine-2-ones can be constructed on a solidsupport from three separate components: 2-aminobenzophenones, α-aminoacids, and (optionally) alkylating agents, as shown in the reactionscheme of FIG. 1.

Preferred 2-aminobenzophenones include the substituted2-aminobenzophenones, for example, the halo-, hydroxy-, andhalo-hydroxy-substituted 2-aminobenzophenones, such as4-halo-4′-hydroxy-2-aminobenzophenones. A preferred substituted2-aminobenzophenone is 4-chloro-4′-hydroxy-2-aminobenzophenone.Preferred α-amino acids include the 20 common naturally occurringα-amino acids as well as α-amino acid mimicking structures, such ashomophenylalanine, homotyrosine, and thyroxine.

Alkylating agents include both activated and inactivated electrophiles,of which a wide variety are well known in the art. Preferred alkylatingagents include the activated electrophiles p-bromobenzyl bromide andt-butyl-bromoacetate.

In the first step of such a synthesis, the 2-aminobenzophenonederivative, (1) of FIG. 1, is attached to a solid support, such as apolystyrene solid support, through either a hydroxy or carboxylic acidfunctional group using well known methods and employing anacid-cleavable linker, such as the commercially available[4-(hydroxymethyl)phenoxy]acetic acid, to yield the supported2-aminobenzophenone, (2). See, for example, Sheppard and Williams,((1982)) Int. J. Peptide Protein Res. 20:451-454). The 2-amino group ofthe aminobenzophenone is preferably protected prior to reaction with thelinking reagent, for example, by reaction with FMOC—Cl(9-fluorenylmethyl chloroformate) to yield the protected amino group2′-NHFMOC.

In the second step, the protected 2-amino group is deprotected (forexample, the —NHFMOC group may be deprotected by treatment withpiperidine in dimethylformamide (DMF)), and the unprotected2-aminobenzophenone is then coupled via an amide linkage to an α-aminoacid (the amino group of which has itself been protected, for example,as an —NHFMOC group) to yield the intermediate (3). Standard activationmethods used for general solid-phase peptide synthesis may be used (suchas the use of carbodiimides and hydroxybentzotriazole orpentafluorophenyl active esters) to facilitate coupling. However, apreferred activation method employs treatment of the 2-aminobenzophenonewith a methylene chloride solution of the of α-N—FMOC-amino acidfluoride in the presence of the acid scavenger4-methyl-2,6-di-tert-butylpyridine yields complete coupling via an amidelinkage. This preferred coupling method has been found to be effectiveeven for unreactive aminobenzophenone derivatives, yielding essentiallycomplete coupling for derivatives possessing both 4-chloro and 3-carboxydeactivating substituents.

In the third step, the protected amino group (which originated with theamino acid) is first deprotected (for example, —NHFMOC may be convertedto —NH₂ with piperidine in DMF), and the deprotected compound is reactedwith acid, for example, 5% acetic acid in DMF at 60° C., to yield thesupported 1,4-benzodiazepine derivative, (4). Complete cyclization hasbeen reported using this method for a variety of 2-aminobenzophenonederivatives with widely differing steric and electronic properties.

In an optional fourth step, the 1,4-benzodiazepine derivative may bealkylated, by reaction with a suitable alkylating agent and a base, toyield the supported fully derivatized 1,4-benzodiazepine, (5). Standardalkylation methods, for example, an excess of a strong base such as LDA(lithium diisopropylamide) or NaH, may be used; however, such methodsmay result in undesired deprotonation of other acidic functionalitiesand over-alkylation. Preferred bases, which may prevent over-alkylationof the benzodiazepine derivatives (for example, those with ester andcarbamate functionalities), are those which are basic enough tocompletely deprotonate the anilide functional group, but not basicenough to deprotonate amide, carbamate or ester functional groups. Anexample of such a base is lithiated 5-(phenylmethyl)-2-oxaxolidinone,which may be reacted with the 1,4-benzodiazepine in tetrahydrofuran(THF) at −78° C. Following deprotonation, a suitable alkylating agent,as described above, is added.

In the final step, the fully derivatized 1,4-benzodiazepine, (6), iscleaved from the solid support. This may be achieved (along withconcomitant removal of acid-labile protecting groups), for example, byexposure to a suitable acid, such as a mixture of trifluoroacetic acid,water, and dimethylsulfide (85:5:10, by volume). Alternatively, theabove benzodiazepines can be prepared in soluble phase. The syntheticmethodology was outlined by Gordon et al. (1994) J Med. Chem.37:1386-1401, which is hereby incorporated by reference. Briefly, themethodology comprises trans-imidating an amino acid resin withappropriately substituted 2-aminobenzophenone imines to form resin-boundimines. These imines can be cyclized and tethered by procedures similarto those in solid-phase synthesis described above. The general purity ofbenzodiazepines prepared using the above methodology can be about 90% orhigher.

2. Preparation of 1,4-benzodiazepine-2,5-diones

A general method for the solid-phase synthesis of1,4-benzodiazepine-2,5-diones has been reported in detail by Boojamra,C. J. et al. (1996) J. Org. Chem. 62:1240-1256. This method can be usedto prepare the compounds of the present invention. This method isdepicted in FIG. 2.

Briefly, a Merrifield resin, for example, a (chloromethyl)polystyrene isderivatized by alkylation with 4-hydroxy-2,6-dimethoxybenzaldehydesodium (3) to provide resin-bound aldehyde (4). An α-amino ester is thenattached to the derivatized support by reductive amination usingNaBH(OAc)₃ in 1% acetic acid in DMF. This reductive amination results inthe formation of a resin-bound secondary amine (5).

The secondary amine (5) is acylated with a wide variety of unprotectedanthranilic acids result in support-bound tertiary amides (6). Thisacylation can be best achieved by performing the coupling reaction inthe presence of a carbodiimide and the hydrochloride salt of a tertiaryamine. One good such coupling agent is1-ethyl-8-[8-(dimethylamino)propyl]carbodiimide hydrochloride. Thereaction is typically performed in the presence of anhydrous1-methyl-2-pyrrolidinone. The coupling procedure can be repeated oncemore to ensure complete acylation.

Cyclization of the acyl derivative (6) can be accomplished throughbase-catalyzed lactamation through the formation of an anilide anion (7)which would react with an alkylhalide for simultaneous introduction ofthe substituent at the 1-position on the nitrogen of the heterocyclicring of the benzodiazepine. The lithium salt of acetanilide is a goodbase to catalyze the reaction. Thus, (6) can be reacted with lithiumacetanilide in DMF/THF (1:1) for 30 hours followed by reaction withappropriate alkylating agent provides the fully derivatizedsupport-bound benzodiazepine (8). The compounds (1) can be cleaved fromthe support in good yield and high purity by using TFA/DMS/H₂O (90:5:5).

Some examples of the α-amino ester starting materials, alkylatingagents, and anthranilic acid derivatives that can be used in the presentinvention are listed by Boojamra, supra at 1246; Additional reagents canbe readily determined and either can be commercially obtained or readilyprepared by one of ordinary skill in the art to arrive at the novelsubstituents disclosed in the present invention.

For example, from FIG. 2, and from Boojamra, supra, one realizes that:alkylating agents provide the R₁ substituents; α-amino ester startingmaterials provide the R₂ substituents, and anthranilic acids provide theR₄ substituents. By employing these starting materials that areappropriately substituted, one arrives at the desired1,4-benzodiazepine-2,5-dione. The R₃ substituent can be obtained byappropriately substituting the amine of the α-aminoester startingmaterial. If steric crowding becomes a problem, the R₃ substituent canbe attached through conventional methods after the1,4-benzodiazepine-2,5-dione is isolated.

3. Chirality

It should be recognized that many of the benzodiazepines of the presentinvention can exist as optical isomers due to chirality wherein thestereocenter is introduced by the α-amino acid and its ester startingmaterials. The above-described general procedure preserves the chiralityof the α-amino acid or ester starting materials. In many cases suchpreservation of chirality is desirable. However, when the desiredoptical isomer of the α-amino acid or ester starting material isunavailable or expensive, a racemic mixture can be produced which can beseparated into the corresponding optical isomers and the desiredbenzodiazepine enantiomer can be isolated.

For example, in the case of the 2,5-dione compounds, Boojamra, supra,discloses that complete racemization can be accomplished bypreequilibrating the hydrochloride salt of the enantiomerically pureα-amino ester starting material with 0.3 equivalents of i-Pr₂EtN and theresin-bound aldehyde for 6 hours before the addition of NaBH(OAc)₃. Therest of the above-described synthetic procedure remains the same.Similar steps can be employed, if needed, in the case of the1,4-benzodiazepine-2-dione compounds as well.

Methods to prepare individual benzodiazepines are well-known in the art.See for example, U.S. Pat. Nos. 3,415,814; 3,384,635; and 3,261,828,which are hereby incorporated by reference. By selecting theappropriately substituted starting materials in any of theabove-described methods, the benzodiazepines of this invention can beprepared with relative ease.

From the above description of the invention, one of skill in the artreadily understands that the various methods of treatment, diagnosticmethods, use of compounds to prepare medicaments, delivery of suchmedicaments, and the making of the compounds, can be practiced in manydifferent ways. It is also understood that preferred features andcharacteristics of one aspect of the invention are applicable to anyother aspect of the invention. In addition, the disclosure of thisinvention, coupled with the ordinary skill in the art can lead tofurther treatments, diagnostic methods, uses, formulations andcompositions. Such further treatments, diagnostic methods uses,formulations, and compositions are within the scope of this invention,which scope is thus considerably broader than the many examplespresented herein, including the ones presented below.

EXAMPLES

General Methods

Cell Preparation

Cell lines were cultured in complete media (RPMI or DMEM containing 10%fetal bovine serum supplemented with penicillin, streptomycin, andL-glutamine) at 37° C., 5% CO₂. For activity assays, cells in log-phasegrowth were removed and diluted to a concentration between 100,000 and300,000/mL. Some cells were kept in complete media, while an identicalaliquot was exchanged into reduced serum media (RPMI or DMEM containing0.2% fetal bovine serum supplemented with penicillin, streptomycin, andL-glutamine) by centrifugation.

Activity Assay

Cells in both complete media and reduced serum media were dispensed into96 well plates in 100 μL aliquots giving 10,000 to 30,000 cells/well.Compound was then added to appropriate wells in the plate (2 μL of each50×stock) at concentrations between 1 M to 20 μM. Cells were thencultured overnight 37° C., 5% CO₂). Relative cell number/cell viabilitywas measured using standard techniques (trypan blueexclusion/hemocytometry, MTT dye conversion assay).

Example 1 Ability to Induce Cell Death

Several individual representative compounds that induce apoptosis havebeen shown above. Of these, the most potent compound is identified asCompound 1, which is shown below.

Compound 1 was used to induce cell death in a variety of cells by usingthe above-described materials and methods. Table 1 shows cell viabilitydata after 18 hours of culture with Compound 1 in reduced serum media asdescribed above. TABLE 1 4 μM Cell Line Source Type Bz 6 μM Bz 10 μM BzJurkat human T-cell 0 0 0 IMR human neuroblastoma 100 0 0 SHSY-5Y humanneuroblastoma Nd 0 0 Shep human neuroblastoma Nd 80 0 293T humanEmbryonic Nd Nd 30 fibroblast RAW 246.7 murine monocytic 70% 0 0 NIH 3T3murine fibroblast Nd Nd 25(lower numbers equal increased killing).Nd = Not determined

Example 2

MRL/MpJ-lpr/lpr (MRL-lpr) mice develop similar serological andhistological manifestations of autoimmune disease as human SLE. Thesemice were developed by a series of cross-breeding of inbred strainsuntil an autoimmune phenotype appeared. (Theofilopoulos A. N. and DixonF. J. (1985) Adv. Immunol. 37:269-390). The MRL-lpr mice arecharacterized by the spontaneous development of systemic autoimmunedisease. This disease is manifested in several physiological locationsand resembles a variety of human diseases. For instance, the kidneydamage in these mice is associated with high serological titers ofanti-DNA as in human SLE. They also develop an erosive arthropathy and alymphocytic infiltration of the salivary glands, similar to the humandiseases rheumatoid arthritis (RA) and Sjörgen's disease, respectively(Theofilopoulos, supra).

In general MRL-lpr mice have a profound defect in apoptosis due themutation of the lpr gene locus. (Sakata K. et al. (1998) Clin. Immunol.Immunopathol. 87:1-7). The defect has been linked to a mutation in theFas receptor gene, important in the signaling of apoptosis in activatedlymphocytes. (Watanabe-Fukunaga, R. et al. (1992) Nature 356:314-317).Consequently, these mice show a profound lymphoproliferation resultingin massive enlargement of the lymph nodes and spleen. Grossly, thesemice demonstrate swollen footpads and erythematous skin lesions.Histologically, glomerulonephritis, arthritis, and inflammatoryinfiltration of the salivary glands are notable.

Methods

Mice

Six week old, female MRL-lpr mice were purchased from JacksonLaboratories (Bal Harbor, ME). The animals were allowed to adapt totheir environment for one week prior to commencement of the treatmentstudy. The mice were housed in a climate controlled specificpathogen-free environment on a 12 hour light dark cycle with food andwater ad libitum. Once a week, weights were measured and proteinuria wasexamined using a colorimetric reaction (Boehringer Mannheim ChemStrip6).

Treatment Regimen

Mice were randomized into three groups: controls receiving PBS (50 μL,qod), controls receiving DMSO (50 μL, qod), and mice receiving Compound1 in 50 μL of DMSO (60 mg/kg qod ip for 20 mice and 30 mg/kg qod ip for10 mice). Intraperitoneal injections were given with a 30 G needle andglass syringes (Hamilton) on an every other day dosing schedule.Treatment started at 7 weeks of age for the control mice (thosereceiving PBS and DMSO) and at 8 or 9 weeks for the treatment mice. Atthe end of the study, blood was collected by tail bleeds. The mice weresubsequently anesthetized by metophane inhalation and were sacrificed byexsanguination by axillary dissection. Sample organs were then removedfor histological analysis.

Statistical Analysis

Analysis of statistical significance was done using the computer programSPSS. Unless otherwise noted, the Mann-Whitney U test (one-tailed) wasused and probability values >5% were considered insignificant.

Results

Disease Progression

MRL-lpr mice are known to develop a kidney disease very similar to thatseen in the human autoimmune disease Systemic Lupus Erythematosus (SLE).The hallmark of this disease is a glomerulonephritis that results inloss of kidney function and eventual death due to kidney failure. Amarker for the development of kidney disease is the amount of proteinpresent in the urine. As kidney function deteriorates, the glomerularfiltration mechanism fails and proteinuria increases. Unlike theperiodicity of the human disease, the murine form of lupus isprogressive; thus, once a mouse develops nephritis and the ensuingproteinuria the disease progresses on a continuum until death. Thisallows the use of proteinuria measurements to follow the progression ofkidney disease in the MRL-lpr mice.

The development of disease in our study was followed by weeklymeasurement of the proteinuria. Any given mouse was determined to havelupus if she had proteinuria values >2+(>100 mg/dL) for 2 or moreconsecutive weeks. No mice meeting this criterion were ever found tohave drops in proteinuria below 2+. Furthermore, the mice that diedwith >2+proteinuria were found to have very significantglomerulonephritis and the mice that died with values <2+had healthykidneys and causes of death unrelated to the autoimmune disease.

FIG. 3 provides disease progression analysis for mice treated 60 mg/kgqod. Similar data are obtained with the 30 mg/kg qod dosing schedule. Asseen in FIG. 3, treating mice with Compound 1 significantly delays theonset of and effectively treats the lupus-like disease relative to thecontrol mice (p=0.0043). These data are supported by the observationthat BUN values for the treated animals are normal whereas thosereceiving vehicle alone are in renal distress.

Laboratory Diagnostics

The blood from the animals was analyzed for alterations in total numbersof white blood cells (WBC) as well as differential representation ofsubtypes. Mice receiving Compound 1 (60 mg/kg) have nearly identicalvalues for hematocrit, platelet count, and WBC relative to thosereceiving vehicle alone (Table 2). TABLE 2 Complete Blood Count withDifferential (mean ± standard deviation). Group HCT PLAT WBC POLYSLYMPHS MONO EOS BASO Control 46.70 809 13.22 31.50 65.07 2.35 0.88 0.13(0.91) (129) (4.65) (8.38) (9.61) (2.03) (0.42) (0.06) Treatment 42.60895 13.68 43.00 53.66 2.01 1.15 0.17 (7.57) (148) (7.27) (16.37) (16.81)(1.05) (1.95) (0.27) p value 0.267     0.183 0.5 0.069 0.069 0.473 0.1470.267HCT, hematocrit (%);PLAT, platelets (K/μL);WBC, white blood cells (K/μL);POLYS, polymorphonuclear cells (%);LYMPHS, lymphocytes (%);MONO, monocytes (%);EOS, eosinophils (%);BASO, basophils (%).

Autoantibodies

Serum samples from all of the mice were analyzed to determine the titerof antibodies to several autbantigens (Table 3). These antibodies aretotal serum polyclonal antibodies. At the termination of the study, themice receiving Compound 1 showed significantly lower titers ofantibodies to ssDNA (p=0.019), histones (p=0.0056), and La antigen(p=0.0265). Anti-dsDNA titers were lower in the treatment mice but notstatistically different from those in the control animals (p=0.082).There was also no difference in antibodies to Ro antigen between the twogroups of mice; however, the actual absorbance measurements were verylow for these ELISAs and any differences may have been masked by thesensitivity of the assay. Anti-Sm titers were only observed in a few ofthe animals in both groups and no conclusions could be made regardingdifferences between the groups. These anti-Sm findings are consistentwith the literature, which reports that only 10% of MRL-lpr mice areexpected to be positive for antibodies against Sm antigen (Murphy, E. D.(1981). For data on lymphoproliferation (lpr) and other single-locusmodels for murine lupus, see Immunologic Defects in Laboratory Animals(E. M. Gershwin and B. Merchant, eds.); Vol. 2, pp. 143-173 (Plenum,N.Y.). The observed differences in autoantibody levels are found in abackground of very high total IgG concentrations which do not differstatistically between the control and the treatment groups (p=0.3312).TABLE 3 Autoantibody Titers Anti-ssDNA Anti-dsDNA Anti-Histone* TotalIgG Anti-Ro Anti-La (U/ml) (U/ml) (OD) (mg/ml) (U/ml) (U/ml) Drug 508 ±193 247 ± 101 0.613 ± 0.526 23.3 ± 6.2 304 ± 256 226 ± 162 Group Control887 ± 328 650 ± 454 1.387 ± 0.537 25.8 ± 8.7 456 ± 328 529 ± 462 Group pvalue 0.019 0.082 0.0056 0.3312 0.1588 0.0265*Titers were not available at the time of this report. Anti-histonelevels are reported as OD₄₀₅ values at a 1/400 dilution of serum.

Joint Histology

In addition to the lupus syndrome, MRL-lpr mice spontaneously develop anerosive arthropathy that resembles human rheumatoid arthritis, bothhistologically and serologically. The arthritic lesions in these miceare characterized by inflammatory changes in the synovium and theperiarticular connective tissue, frequently accompanied by the presenceof circulating rheumatoid factors in the serum. This arthritic processis progressive and proceeds through several different stages from a mildsynovitis to an erosive arthritis, which can eventually lead to ascarred joint. Histologically, the majority of 5 month old MRL-lpr micedemonstrate synovial cell proliferation, destruction of articularcartilage and subchondral bone, infiltration of synovial stroma byinflammatory cells, periarticular inflammation (vasculitis, myositis,tendinitis, perineuritis), exudates, pannus formation, and subcutaneousfibrinoid nodules (Hang, L. et al. (1982) J. Exp. Med. 155:1690-1701;Koopman, W. J. and Gay, S. (1988) Scand. J. Rheumatology. Suppl.75:284-289).

The paws of all the mice treated with Compound 1 were examined for signsof arthritis and synovitis. The control mice (those receiving vehiclealone) have a severe synovitis characterized by a marked thickening ofthe synovium with occasional formation of papillary, villousconfigurations. Typically, the synovial pathology was a result ofsynovial cell proliferation and infiltration of the synovial stroma byinflammatory cells. In a substantial percentage of the control mice, thedisease process was accompanied by pannus formation and erosion of thearticular surface (both articular cartilage and subchondral bone). Incontrast, the treatment mice were found to have a milder synovitis aswell as fewer erosions and limited pannus formation (Table 4). Thecharacter of the disease in the animals receiving Compound 1 was muchless aggressive with less synovial cell proliferation and inflammatoryinfiltration. Of further interest, it was observed that the treatmentmice had a lessened degree of periarticular inflammation. Thecombination of these findings suggest that Compound 1 is amelioratingthe arthritic disease process that typically destroys the joints ofMPL-lpr mice. TABLE 4 Synovial Pathology Average Number of Number ofHistologic Mice with Number of Mice with Number Score Synovitis ≧ Micewith Pannus Group of mice Synovitis 2^(†) Erosions^(†) Formation^(†)Control 7 2.1 5 (71%) 4 (57%) 4 (57%) Treatment 7 1.3 0 (0%)  1 (14%) 1(14%) p value p = 0.001 p = 0.01 p = 0.13 p = 0.13^(†)p value determined by cross-tabulation and chi-square analysis.

Delayed Type Hypersensitivity (DTH)

Mice treated with Compound 1 (60 mg/kg) showed no difference in DTHresponse to TNBS on comparison to the control mice (FIG. 4C).Importantly, neither group of animals demonstrated a significant footpadswelling following antigen challenge. This phenomenon has beendocumented and old MRL-lpr mice (>10 weeks) are expected to have adiminished in vivo T cell response to stimulus as evidenced by theabsence of a DTH response. See Okuyarna, H. et al. (1986) Clin. Exp.Immunol. 63:87-94, and Scott, C. F. et al. (1984) J. Immunol.132:633-639. However, suppression of T cells can result in a rescue ofthe DTH response. See, Okuyama, H. et al. (1989) Int. Arch. AllergyAppl. Immunol. 1588:394-401. Such a rescue was not observed in thisstudy's treatment protocol. These data suggest that Compound 1 does notalter T cell function. FIGS. 4A-4C show the results of the FootpadSwelling experiment.

Immune Cell Function

Thymidine uptake assays of using both stimulated and unstimulated T andB cells was conducted to determine if Compound 1 affectslymphoproliferation in vitro. At about a concentration of 10 μM, noeffect on lymphocyte proliferation was observed.

Example 3 Compound 1 as a Lymphotoxic Agent

Methods

Animals and Experimental Design:

Female NZB/W mice (Jackson Labs) were housed in specific pathogen-free,environmentally controlled rooms operated by the University ofMichigan's Unit for Laboratory Animal Medicine with 12 hr light-darkcycles and were given food and water ad libitum. Mice were randomlydistributed into treatment and control groups. All mice were dosedthrough intraperitoneal injections using a Hamilton repeating dispenserwith glass microliter syringes and 30 gauge needles. Control micereceived vehicle (50 μL aqueous DMSO) and treatment mice receivedCompound 1 dissolved in vehicle. Animal weights were determined weekly,and dosing schedules readjusted thereafter.

Collection of Blood Tissues:

Peripheral blood was obtained from the tail veins of all mice forcomplete blood counts analysis and collection of serum. Blood was firstallowed to clot at room temperature for 1 h, and then overnight at 4° C.Serum was separated from the formed clot by centrifugation (6 min.,16,000×g). A section of spleen was removed aseptically for preparationof single cell suspensions. Samples of the following organs werepreserved in 10% buffered-formalin: heart, liver, lung, spleen, kidneysmall intestine, reproductive system, salivary glands, thymus,mesenteric and axillary lymph nodes, and skin. Additional sections ofkidney and spleen were preserved by snap-freezing in OCT. Bone marrowsmears were prepared from each femur.

Histology:

All histological determinations were made in a blinded fashion by apathologist. Formalin-fixed sections were cut and stained withhematoxylin and eosin (H&E) using standard protocols (Luna, L. G., in:Manual of Histological Staining Methods of the Armed Forces Institute ofPathology, McGraw-Hill, N.Y. (1960)). Immune-complex deposition in thekidneys was evaluated by direct immunofluorescence using frozen sectionsstained with FITC-conjugated goat anti-mouse IgG (Southern BiotechnologyAssociates, Birmingham, Ala.) and C3 (Cappel-Organon Teknika, Durham,N.C.). The degree of lymphoid hyperplasia was scored 0-4+scale.

TUNEL Staining:

Frozen spleen sections (4 pm thick) were assayed for DNA strand breaksusing the In Situ Cell Death Detection kit (Roche MolecularBiochemicals) according to the manufacturer's protocols. Sections wereanalyzed using a 0-4+ scale. Sections were blindly evaluated andassigned a score on the basis of the amount of TUNEL-positive staining.

Fluorescence Analysis of Lymphocyte Populations:

Single cell suspensions were prepared by teasing apart the spleen inmedia, followed by removal red blood cell with isotonic lysis buffer(Kruisbeek, A. M., in: Current Protocols in Immunology, eds., Coligan,J. E. et al., pp. 3.1.2-3.1.5, John Wiley & Sons, Inc. (1997)). 106cells were stained at 4° C. with fluorescently-conjugated anti-Thy 1.2(Pharmingen, clone: 53-2.1, 1, μg/mL) and/or anti-B220 (Pharmingen,clone: RA3-6B2 1, μg/mL) for 15 min. In samples stained to detectouter-membrane phosphatidyl serine, cells were then incubated withFITC-conjugated Annexin V and PI according to manufacturer protocols(Roche Molecular Biochemicals). Cells were analyzed on a Coulter ELITEflow cytometer. For each sample, at least 10,000 events were counted.

Serum Anti-DNA:

Titers were determined by direct ELISA as previously described (Swanson,P. C. et al., J. Clin. Invest. 97:1748-1760 (1996)). Detection of IgGanti-DNA used an alkaline phosphatase-conjugated Goat anti-Mouse IgG(H-chain only) secondary antibody ( 1/1000 dilution, SIGMA). To convertabsorbance readings into titers, pooled serum from unmanipulated eightmonth old female NZB/W was used as a reference standard which wasarbitrarily assigned a value of 1000 U/mL.

Serum Immunoglobulin:

Concentrations were determined by capture ELISA. Goat anti-Mouse Ig(Southern Biotechnology Associates) was diluted to 10 pg/mL in PBS andcoated overnight at 4° C. on Immulon II microtiter plates. Otherwise,ELISAs were performed as previously described. To convert absorbancesinto concentrations, a standard curve was generated using a previouslyquantified mouse immunoglobulin reference serum (ICN Biomedicals,Aurora, Ohio).

Blood Urea Nitrogen (BUN) and Complete Blood Counts (CBC):

Serum BUN measurements were conducted by the University of MichiganHospital's clinical pathology laboratory. CBC analyses were conducted bythe diagnostic laboratory of University of Michigan's Unit forLaboratory Animal Medicine. Automated counts determined by a Hemavet 15OR were confirmed by visual examination of blood smears.

Serum 1,4-benzodiazepine Levels:

Serum samples from mice injected with Compound 1 were precipitated withacetone (5× volume, −20° C., 10 min). After centrifugation (16000×g, 10min), the supernatant containing Compound 1 was concentrated in vacuo,and then extracted from any remaining protein using a Sep-pak C18 column(Waters Corp.) running a step gradient from 10% acetonitrile in water to100% acetonitrile. Material eluting in the organic fraction wasconcentrated in vacuo, and then analyzed by reversed-phase HPLC using aPhenomenex C18 column. Peak areas were determined using a Shimadzuintegrator and were referenced to a standard curve.

Statistical Analysis:

Statistical analyses was conducted using the SPSS software package. TheMann-Whitney U and chi-square tests were used for histological andclinical data. Student's t-test was used for flow cytometric data.Correlations were assessed by ANOVA.

Example 4

A series of experiments were conducted to characterize the nature ofcell death caused by Compound 1 in terms that could point toward thedeath mechanism. Compound 1 treated cells stained positive in TUNELassays suggesting treatment resulted in free-ends of DNA (Gorczyca, W.et al., Leukemia 7:659-670 (1993)), a feature of apoptosis. Caspaseactivity and mitochondrial permeability are two central components ofthe intracellular death machinery (See, Zamzami, N. et al., J. Exp. Med.183:1533-1544 (1996) and Los, M. et al., Immunity 10:629-639 (1999)).Cyclosporin A (CsA) regulates the mitochondrial permeability transition(MPT) (Zoratti, M. and Szabo, I., Biochim. Biophys. Acta 1241:139-176(1995)), protecting cells from death pathways that rely on the releaseof apoptogenic factors from the mitochondria. CsA provided dramaticprotection against Compound 1, such that 90% of the cells that wouldhave been killed by Compound 1 survive when CsA (10 μM) is added to theculture (FIG. 6B). Pretreating cells with z-VAD-fmk (20 pM), anon-specific caspase inhibitor (Garcia-Calvo, M. et al., J. Biol. Chem.273:32608-32613 (1998)), protects cells to a lesser extent: viability inz-VAD-fmk pretreated cells is 25% greater than cells treated withCompound 1 alone. Together, these data suggest that Compound 1-induceddeath of NZB/W lymphocytes requires the MPT while caspase activation isof secondary importance.

To determine if the effects on lymphocytes in vitro also occur in vivo,8-month old female NZB/W mice were dosed with Compound 1 (60 mg/kg IPdaily). This dose results in peak serum concentrations of 10 μM (1 hpost-injection) with trough levels of 1-2 μM (18-24 h post-injection).After 7 days, mice were sacrificed and their spleen cells examined byflow cytometry. Splenocyte viability, measured on the basis of PIexclusion, was significantly decreased in treated versus controlinjected animals as shown in Table 5, below. TABLE 5 Apoptotic ApoptoticCell Viability B-cells T-cells (PI⁻) (B220⁺, Thy1.2⁻) (B220⁻, Thy1.2⁺)Control 85 ± 2 25 ± 5 12 ± 4 Bz-423 81 ± 2 32 ± 6 12 ± 9 p = 0.003 p =0.02 p = 0.50Flow cytometric measurements of splenocytes after in vivo treatment.

In this experiment, 8 month old NZB/W mice were divided into treatmentand control groups of seven mice each. Treatment mice were dosed withCompound 1 (60 m kg qd) and the controls received vehicle (50 μL DMSOqd) for 7 days. The overall viability of the splenocytes isolated fromeach mouse was determined on the basis of PI exclusion. The decrease inviability is smaller than that observed after in vitro treatment becauseintervening processes (i.e., cell recruitment, proliferation andclearance of early apoptotic cells by the reticuloendothelial system(RES)) decrease detectable splenocyte death. Apoptotic cells are shownas a percentage of PI-excluding B- and T-cells. Mixed splenocytes wereisolated from treated animals and then placed in culture briefly (2×106cells/mL for 3 h in RPMI containing 10% FBS) to allow expression of theapoptotic phenotype in the absence of RES clearance. Subsequently, cellswere stained with Annexin V-FITC,. PI and monoclonal antibodies specificfor either Thy-1.2 or B 20. Each value is based on the flow cytometricanalysis of 20,000 viable cells.

Treatment of NZB/W Mice with Compound 1

At therapeutic doses, current treatments for organ-threatening lupusseverely deplete all lymphoid cells, resulting in serious side effectsassociated with immunosuppression (Donadio, J. V. and Glassock, R. J.,Am. J. Kidney Dis. 21:239-250 (1993)). The B cell selectivity coupledwith the moderate overall effect on splenic lymphocyte viabilitydistinguish Compound 1 from these treatments. Hence, Compound 1represents a lead compound in a new class of lymphotoxic agents possiblywith a favorable benefit-toxicity profile. To determine the effect ofCompound 1 on autoimmune nephritis and how long-term dosing affects theimmune system, a longitudinal treatment study was conducted using femaleNZB/W mice.

In this study, mice received intraperitoneal injections of Compound 1(n=25; 60 mg/kg), or vehicle (n=20) every other day over 3 mo. from 6.5to 9.5 mo. of age. This time span represents a point after the onset ofglomerulonephritis and continues through the 50% mortality point(Theofilopoulos, A. N. and Dixon, F. J., Adv. Immunol. 37:269-390(1985)). Mice were sacrificed periodically and examined for the impactof Compound 1 on nephritis and the peripheral immune system.

Microscopic examination of kidney tissue reveals that treatment withCompound 1 is protective throughout the course of treatment. Kidneysections taken from control mice indicate a diffuse, proliferativeglomerulonephritis with proliferation of all cellular elements andoccasional wire-loop formation consistent with an averagehistopathologic score of 3+ (FIGS. 6 and 7). In contrast, treatment micehave much milder changes with less cellular proliferation and wire-loopformation and a score of 1+ (p<0.002). As seen in FIG. 6, administrationof Compound 1 reduced both glomerular IgG and C3 deposition throughoutthe course of treatment (IgG, p<0.003; C3, p<0.04). The degree ofdeposition within an individual mouse strongly correlates with itshistopathologic score: mice with reduced Ig deposition show lessglomerular injury (p<0.002). Mechanistically, this finding is consistentwith lymphoid cell death in vivo, presumably leading to fewer(pathogenic) B cells.

The histological differences observed between the treatment and controlgroups were confirmed by clinical measures of kidney function. At thetime of sacrifice, 85% of control mice have abnormally high blood ureanitrogen (BUN) levels (2 30 mg/dL) (Gordon, C. et al., Clin. Immunol.Immunopath. 52:421-434 (1989)), while 31% of treatment mice haveelevated BUN (p<0.007). Similarly, 39% of control mice have significantproteinuria (>100 mg/dL) (Adelman, N. E. et al., J. Exp. Med.158:1350-1355 (1983)), while only 18% treatment mice developed thisfinding (p<0.11). For each mouse, the histological score is stronglycorrelated with both the proteinuria and BUN levels (p<0.002); the micewith less severe disease have lower proteinuria and BUN titers. Thesedata confirm that the histological measures of nephritis reflect thefunctional status of the kidneys. Together, these findings indicate thatCompound 1 protects NZB/W mice from both the progression and the effectsof autoimmune glomerulonephritis.

Compound 1 Reduces B Cell Number

NZB/W mice develop disease-related lymphoid hyperplasia marked by anexpansion of B-cells that coincides with the increase in serumautoantibody titers (See, Theofilopoulos, A. N. and Dixon, F. J., Adv.Immunol. 37:269-390 (1985) and Ermak, T. H. et al., Lab. Invest.61:447456 (1989)). Spleens from control mice reflect this phenomenon: anexpansion of the white pulp is observed causing both distortion ofnormal spleen architecture and a reduction in the red pulp (FIG. 10).The mice that received Compound 1 have a more normal balance of red andwhite pulp. Scoring multiple spleen sections from all treatment andcontrol animals revealed a statistically significant reduction oflymphoid hyperplasia in the treatment group (2+ vs. 3+ on a 0-4+ scale;p<0.02).

The constitution of the splenic lymphoid cell compartment after 6 and 12wk of treatment was analyzed by flow cytometry. As shown in Table 6,Compound 1 reduces the B cell fraction by 10-15% relative to controlmice (p<0.05), whereas the fraction of T-cells remains unaffected bytreatment. TABLE 6 Timepoint Control B cells (B220⁺Thy1.2⁻) T-cells(B220⁻Thy1.2⁺)  6 week Control 58 ± 5 18 ± 6 Bz-423 49 ± 5 26 ± 5 p =0.04 p = 0.10 12 week Control 67 ± 5 17 ± 5 Bz-423 59 ± 6 16 ± 4 p =0.04 p = 0.40Flow cytometric determination of splenic lymphocyte populations presentafter longitudinal treatment.

This difference can be explained either by an effect on lymphocyteproliferation or by the selective reduction of lymphocytes. SinceCompound 1 does not alter splenocyte proliferation in vitro, we favorthe later explanation. That no significant increase in the fraction of Tcells is observed, suggests killing of T cells occurs to a lesser extentthan killing of B cells, which is consistent with the findings from thein vitro studies and short term dosing experiments presented above.

To directly determine the effect of Compound 1 on lymphoid cell death invivo, splenic tissue was analyzed for evidence of cell death using theTUNEL reaction. In sections from 10 representative control mice,TUNEL-positive cells amount to less than 1% of the total number of cellsand the staining was randomly dispersed throughout the section. Bycontrast, mice treated with Compound 1 possess up to a 5-fold increasein numbers of TUNEL-positive cells (ca. 3+ staining) in 50% of thesections (p<0.05). Unlike the sections from the control mice, theTUNEL-positive cells in these sections show prominent clustering (FIG.10). Combined TUNEL analysis and immunohistochemical staining for aB-cell surface marker (B220) indicate that cells demonstratingdrug-induced TUNEL positivity are B-cells (FIG. 10).

The action of Compound 1 against B cells and the improvement inautoantibody-mediated renal disease predicts a decrease in autoantibodytiters. Serum obtained during the course of treatment was assayed fortotal IgG as well as anti-dsDNA. Total immunoglobulin levels are notaltered over the course of treatment (e.g., after 9 weeks of treatment,control=4.0±2.2 mg/mL; Compound 1=4.0+1.3 mg/mL, p>0.4). In contrast,anti-dsDNA titers are reduced after 3 weeks of treatment with Compound 1(control=733+546 U/mL; Compound 1=496+513 U/mL, p<0.06). However, byabout 9 weeks of treatment anti-dsDNA levels in treated mice areindistinguishable from controls (control=812+695 U/mL; Compound1=944+546 U/mL, p>0.3). The early drop in anti-dsDNA in the context ofunchanged total IgG coupled with the observation that glomerular Igdeposition is reduced throughout the entire course of treatment,suggests that pathogenic cells may have a enhanced sensitivity toCompound 1.

Given the progressive nature of the disease in NZB/W mice, it is likelythat the eventual rise of anti-dsDNA corresponds to a pointcharacterized by increased B-cell proliferation, for example, as can beeseen in Table 6. This hypothesis is also supported by the observationthat administering twice the dose of compound results in a sustainedreduction of anti-dsDNA with disease improvement roughly equivalent tothat seen with the lower dosing.

In summary, these observations link treatment with Compound 1 to diseaseimprovement, as reflected by renal histology, renal function,autoantibody, and C3 deposition, and provides evidence that thistherapeutic effect coincides with the ability of the compound toselectively induce cell death in B-cells.

Compound 1 is not Toxic

Animal tissues were examined for evidence of toxicities commonlyassociated with cytotoxic drugs. Compound 1 does not affect gross animalweight and mice receiving Compound 1 do not have altered groomingpractices or noticeably different behaviors. Microscopic examination ofthe heart, liver, lung, salivary glands, small intestine, and uterusdemonstrated no significant architectural differences between controland treatment groups. Further comparing the control and treatment groupsrevealed that Compound 1 did not increase the number of apoptotic cellsin the non-lymphoid organs. No evidence of increased pneumonitis wasseen in the lungs of the treated animals which is an important indicatorof serious infection commonly observed in animal studies using cytotoxicdrugs (Horowitz, R. E. et al., Lab. Invest. 21:199-206 (1969) and Hahn,B. H. et al., Arthritis Rheum. 18:145-152 (1975)). The lack of excesspneumonitis in treated animals is strong evidence against a significantimmunosuppressive effect. Examination of bone marrow smears revealed nodifferences between treatment and control animals with respect tooverall cellularity or in the proportional representation of specificmyeloid and erythroid precursors. Peripheral blood counts anddifferential white blood cell analysis revealed no decreases inplatelets, granulocytes, lymphocytes, or hematocrit. In aggregate, noevidence of generalized immune suppression or other toxicities weredetected in mice treated with Compound 1.

Example 5

To model neuroblastoma in mice, the mice were transfected with the humanneuroblastoma cell line SKNAS, to cause the cells to overexpress theneuroblastoma associated human oncogene N-myc. The resulting cell lineis designated as D2. These cells form tumors when xenografted into Tcell-deficient athymic mice; thus providing a relevant animal model ofhuman neuroblastoma.

In vitro testing of the D2 cells was conducted to determine theirsensitivity to benzodiazepine. D2 cells were plated into 96-well tissueculture plates at a density of 10,000 cells per well in culture media(DMEM, 10% V:V heat inactivated fetal bovine serum (FBS), 100 μmlpenicillin, 100 μml streptomycin, 290 μml glutamine) and cultured (37°C., 5% CO₂) overnight. Subsequently, culture media was exchanged withmedia containing 1% FBS. Solvent control (dimethyl sulfoxide (DMSO);final concentration 1% V/V) or benzodiazepine at concentrations of2.5-20 μM was added. After 18 hours cell viability was assessed usingthe MTT assay as previously described in this application. FIG. 11demonstrates that benzodiazepine kills D2 cells in a dose-responsefashion.

To test the effect of benzodiazepine on neuroblastoma tumor growth,1×10⁷ D2 cells were aseptically inoculated into the thigh musculature ofeach of eight six-week old nu/nu female mice (Jackson Labs). Beginningone week after tumor cell inoculation, 4 mice were dosed with DMSO (20μl injected into the peritoneal cavity every day) and 4 mice were dosedwith benzodiazepine (2.5 mg dissolved in 20 μl DMSO injected in theperitoneal cavity every day). The mice were evaluated regularly fortumor development and once present the size of the primary tumor wasmeasured every other day. Table 7 demonstrates that in mice that formedtumors, treatment with benzodiazepine significantly decreased the rateof tumor growth. TABLE 7 Treatment and control mice Days for tumorvolume with tumors to increase 5 fold Mouse 1 with Compound 1 9 Mouse 2with Compound 1 12 Mouse 3 with Compound 1 9 Mouse 4 with Compound 1 16Mouse 5 with DMSO 2 3 Mouse 6 with DMSO 3 5Administration of Benzodiazepine slows rate of neuroblastoma tumorgrowth in nu/nu mice (p < 0.02).

Specifically, tumors in control mice increased in volume 5-fold over anaverage 4 day period, whereas 12 days were required for the sameincrease in tumor size in benzodiazepine-treated animals (p<0.02). Thesefindings support the claim that benzodiazepine is able to treat humanmalignant disease in a mouse model. Further, benzodiazepine has specificactivity against human neuroblastoma both in vitro and in vivo.

Example 6

In another line of experiments we sought to determine if benzodiazepineis able to kill tumor cells that are otherwise resistant to presentstandard chemotherapy drugs. Ovarian cancer provides an excellent modelfor studying the problem of chemoresistance in that treatment failuresare commonly ascribed to the emergence of chemotherapy resistant cells.The A2780 human ovarian cancer cell line is known to contain wild-typep53; express low levels of bcl-2 and bcl-x_(L) survival factors; and issensitive to treatment with cis-platinum(II) diamine dichloride (CDDP),a standard chemotherapeutic for treatment of ovarian cancer. These cellswere transfected with an expression vector encoding human bcl-x_(L), asurvival factor that when over-expressed is linked to the development ofchemotherapy resistance. These transfected cells are designated 2B1, andthe empty vector transfected controls are designated vector only. Athird ovarian cancer cell line, designated SKOV3, was also obtained.This cell line is characterized as: 1. Deficient in wild-type p53expression; 2. Expressing high levels of endogenous bcl-x_(L); and 3.Relatively resistant to the cytotoxic actions of CDDP.

Each of these cell lines was maintained using standard tissue cultureconditions in complete media composed of RPMI, 10% FBS, 100 U/mlpenicillin, 100 μ/ml streptomycin, 290 μ/ml glutamine. Each cell typewas plated into a series of separate wells on 24-well tissue cultureplates at 50,000 cells per well. Approximately 24 hours after plating,media was exchanged to contain the same culture media made with only 2%FBS. At this point either control solvent (DMSO, 1% V/V), increasingconcentrations of Compound 1 (4-20 μM), or increasing concentrations ofCDDP (6.7-66.7 μM) was added to cells. After twenty-four hours ofculture all cells present in each well were removed using trypsin-EDTAand mixed with propidium iodide (final concentration 1 μ/ml). Afterincubating 20 minutes cells were analyzed by flow cytometry (CoulterFACS Calibur) to determine cell death on the basis of plasma membraneintegrity measured as the fraction of cells that had taken-up propidiumiodide. FIG. 12 demonstrates that the predicted pattern ofchemosensitivity and resistance towards CDDP (A2780 and vectorsensitive; 2B1 and SKOV3 resistant) was observed. FIG. 13 demonstratesthat benzodiazepine kills each of these types, irrespective of CDDPresistance. Further, benzodiazepine kills ovarian cancer cells that areresistant to standard chemotherapy. Further, benzodiazepine kills tumorcells that express high levels of survival factors (bcl-x_(L)), as wellas those that are deficient in p53 expression.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1-129. (canceled)
 130. A method of treating cancer, comprisingadministering to a subject an effective amount of a benzodiazepinecompound having the structure:


131. The method of claim 130, further comprising co-administering one ormore additional agents to the subject.
 132. The method of claim 131,wherein the additional agent is a chemotherapeutic agent or radiation.133. The method of claim 130, wherein the compound is administeredorally, parenterally, topically or intranasally.
 134. The method ofclaim 130, wherein said cancer comprises neuroblastoma cells.
 135. Themethod of claim 136, wherein said treating comprises inhibiting thegrowth rate of said neuroblastoma cells.
 136. A method of treatinggraft-versus-host-disease, comprising administering to a subject aneffective amount of a benzodiazepine compound having the structure:


137. The method of claim 136, further comprising co-administering one ormore additional agents to the subject.
 138. The method of claim 137,wherein said one or more additional agents is selected from the groupconsisting of immunosuppressive agents, antimetabolite and/orchemotherapeutic agents, antibody and/or Ig agents, immunomodulatingagents, and photoactive agents.
 139. The method of claim 138, whereinsaid immunosuppressive/cytotoxic agents are selected from the groupconsisting of methylprednisolone, prednisone, cyclosporin, sirolimus,tacrolimus, and mycophenolate mofetil.
 140. The method of claim 138,wherein said antimetabolite and/or chemotherapeutic agents are selectedfrom the group consisting of methotrexate, and azathioprine.
 141. Themethod of claim 138, wherein said antibody and/or Ig agents is selectedfrom the group consisting of antithymocyte globulin, IVIG, alemtuzumaband muromonab-CD3.
 142. The method of claim 138, wherein saidimmunomodulating agents comprise thalidomide.
 143. The method of claim138, wherein said photoactive agents comprise methoxsalen.
 144. Themethod of claim 138, wherein the compound is administered orally,parenterally, topically or intranasally.
 145. A method of treatingrheumatoid arthritis, comprising administering to a subject an effectiveamount of a benzodiazepine compound having the structure:


146. The method of claim 145, further comprising co-administering one ormore additional agents to the subject.
 147. The method of claim 145,wherein said one or more additional agents is selected from the groupconsisting of nonsteroidal anti-inflammatory drugs, cyclooxygenase-2(COX-2) inhibitors, disease-modifying agents, immunomodulators,glucocorticoids, and interleukin-1 receptor inhibitors.
 148. The methodof claim 147, wherein said nonsteroidal anti-inflammatory drugs compriseibuprofen.
 149. The method of claim 147, wherein said cyclooxygenase-2(COX-2) inhibitors comprise celecoxib.
 150. The method of claim 147,wherein said disease-modifying agents are selected from the groupconsisting of methotrexate, leflunomide, azathioprine, sulfasalazine,infliximab, adalimumab, and etanercept.
 151. The method of claim 147,wherein said immunomodulators comprise abatacept.
 152. The method ofclaim 147, wherein said glucocorticoids comprise prednisone.
 153. Themethod of claim 147, wherein said interleukin-1 receptor inhibitorsanakinra.
 154. The method of claim 145, wherein the compound isadministered orally, parenterally, topically or intranasally.